The Human Microbiome Project originally focused on the microbiome of the nasal and oral cavities, the skin, the gastrointestinal tract, and the urogenital tract. Like these other organs and organ systems, the respiratory tract is continually exposed to the external environment, but virtually nothing is known about the microbiome of the lung. Indeed, the presence of a lung microbiome in normal individuals has yet to be established conclusively. Since the beginning of the HIV epidemic in the 1980's, HIV-infected individuals have been known to be extremely susceptible to pneumonias caused by opportunistic and non-opportunistic organisms. Comparison of the microbiome of HIV-infected and HIV-uninfected individuals, then, should be a productive way to begin to explore changes in the lung microbiome. Because the microbiome may shift subtly (or not so subtly) over time, we have constructed an experimental design in which each research subject can also serve as his/her own control. In specific aim 1, we will employ molecular and advanced culture-based techniques for the characterization of the bacterial microbiota of the lower respiratory tract in HIV-infected and HIV-uninfected individuals longitudinally. We will compare these data with those obtained from the nasal cavity, the oropharynx, and the stomach. We hypothesize that significant differences will be present, even after controlling for clinical factors. In specific aim 2, we will compare data from currently smoking and non-smoking individuals (never- or former-smokers), and correlate these data with measurements of pulmonary function longitudinally. These studies will assist with our understanding of the pathogenesis of early emphysema development in HIV-infected individuals. We hypothesize that the rate of decline in both smoking and non-smoking HIV-infected subjects will exceed the rate measured in smoking and non-smoking HIV-uninfected subjects, and that this rate of decline will correlate with microbial diversity. Furthermore, we anticipate that microbiota data will relate directly to subject reports of sputum production and dyspnea. In specific aim 3, we will compare colonization with Pneumocystis in HIV-infected and HIV- uninfected individuals. This is our proposal for a collaborative pilot study that will involve all funded centers, but will develop in greater detail by the Steering Committee. We will partner with the other funded data collection sites to determine whether Pneumocystis colonization alters the microbiome. RELEVANCE (See instructions): Virtually nothing is known about the microbiome of the normal lung, and so beginning characterization of the microflora of the lung is an area of considerable scientific and clinical relevance. These studies will characterize the microbiome of the lung and will provide insights into the development of lung disease in HIV-infected and HIV-uninfected populations.